A long-term goal of our research is to elucidate molecular mechanisms that control axon guidance in the developing vertebrate central nervous system (CNS). In vertebrates, opposed gradients of dorsal midlinederived chemorepellents and VM-associated chemoattractants direct spinal commissural axons along a circumferential trajectory to the floor plate (FP) at the VM. Upon crossing the VM, most commissural axons turn rostrally into the longitudinal plane and either project alongside the FP and assemble into a medial axon tract or grow into an intermediate region of the spinal cord and compose a more lateral longitudinal tract, but never re-cross the FP. Post-crossing commissural axons lose responsiveness to midline attractants and gain responsiveness to the midline-/ventral spinal cord-associated repellents, 81its1 -3 and Sema3B/3F in vitro. Consistent with these observations, the repulsive Slit receptors, Rob01 /2 are upregulated on decussated segments of these axons, and an interaction between the cytoplasmic domains of Rob01 /2 and the Netrin receptor, DCC, silences Netrin attraction in vitro. EphB receptors are also selectively expressed on postcrossing segments of commissural axons and likely regulate midline guidance through contact-dependent interactions with their repulsive, FP-associated ligand, ephrin-B3. Paradoxically, Neuropilin (Npn) 2, the 8ema3B/3F receptor is expressed on both pre- and post-crossing segments of commissural axons. Whereas these mechanisms/systems presumably underlie a navigational program that controls the guidance of post-crossing commissural axons, it is not known whether Robo mediated silencing operates within commissural axons in vivo, how EphB and Npn2 receptors regulate the guidance of post-crossing commissural axons, and through what mechanisms these axons choose medial, as opposed to lateral, longitudinal tracts. The basic helix-loop-helix (bHLH) proteins Atoh1 , Neurog1 and Neurog2 are expressed by neuronal progenitors that differentiate into genetically distinct d11 , dl2 and dl4 commissural neurons, respectively. Enhancer elements from these genes can direct reporter expression to commissural neurons/axons and have been used to establish in vivo systems for selectively analyzing the guidance of distinct post-crossing commissural axon populations. The proposed studies will employ genetic labeling strategies in chick and mouse embryos, knockout mice, as well as a variety of in vitro assay systems, and exploit mis-expression strategies in chick embryos to determine: 1) whether the upregulation of Rob01/2 and EphB receptors and Robo-mediated silencing of Netrin attraction alters the responsiveness of post-crossing axons, and whether Robo-mediated inactivation of N-cadherin adhesion regulates the positioning of these axons in longitudinal tracts, and 2) whether interactions between Npn2, Sema3B/3F and, possibly, a subset of cell surface proteins/receptors, in vivo, selectively regulates commissural axon guidance on the contralateral side of the FP.